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| United States Patent |
5,618,541
|
|
Quentin-Millet
|
April 8, 1997
|
Vaccine against Neisseria meningitidis infections
Abstract
A vaccinal pharmaceutical composition which comprises, as therapeutic
agents, at least a first and a second molecule capable of binding to human
transferrin; the said first molecule originating from a first strain of N.
meningitidis which possesses a human transferrin receptor in which the
lower molecular weight subunit (Tbp2) is recognised by an antiserum to the
receptor of N. meningitidis strain 2394 (receptor 2394) and is not
recognised by an antiserum to the receptor of N. meningitidis strain 2169
(receptor 2169); and at least a second molecule originating from a second
strain of N. meningitidis which possesses a human transferrin receptor in
which the lower molecular weight subunit (Tbp2) is recognised by an
anti-receptor 2169 antiserum and is not recognised by an anti-receptor
2394 antiserum.
| Inventors:
|
Quentin-Millet; Marie-Jose (Villeurbanne, FR)
|
| Assignee:
|
Pasteau Merieux Serums et Vaccins (Lyon, FR)
|
| Appl. No.:
|
066167 |
| Filed:
|
June 2, 1993 |
| PCT Filed:
|
September 29, 1992
|
| PCT NO:
|
PCT/FR92/00905
|
| 371 Date:
|
June 2, 1993
|
| 102(e) Date:
|
June 2, 1993
|
| PCT PUB.NO.:
|
WO93/06861 |
| PCT PUB. Date:
|
April 15, 1993 |
Foreign Application Priority Data
| Current U.S. Class: |
424/250.1; 424/249.1; 435/871 |
| Intern'l Class: |
A61K 031/075 |
| Field of Search: |
424/92,249.1,250.1,192
435/871
530/400
|
References Cited
| Foreign Patent Documents |
| 87/02678 | May., 1987 | WO.
| |
| 90/12591 | Jan., 1990 | WO.
| |
Other References
N. Banerjee-Bhatnagar and Carle E. Frasch, "Expression of Neisseria
meningitidis Iron-Regulated Outer Membrane Proteins, Including 1
70-Kilodalton Transferrin Receptor, and Their Potential for Use as
Vaccines," Infection and Immunity, vol. 58, No. 9, Sep. 1990, pp.
2875-2881.
Anthony B. Schryvers and Leigh J. Morris, "Identification and
Characterization of the Human Lactoferrin-Binding Protein from Neisseria
meningitidis," Infection and Immunity, vol. 56, May 1988, pp. 1144-1149.
Griffiths et al; FEMS Microbiology Letters 69:31-36, 1990.
Saukkonen et al; Vaccine 7:325-328, 1989.
Schryvers et al; Can. J. Microbiol 35:409-415, 1989.
Mackinnen et al, Microbiol Pathogenesis 12:415-420, 1992.
Sacchi et al Vaccine 13(1):112-118, 1995.
Zollinger et al, Trans. Royal Soc. Trop. Med Hyg. 85 Suppl. 1 37-43, 1991.
Zollinger et al, Infect. Immun. 40(1):257-264, 1983.
|
Primary Examiner: Housel; James C.
Assistant Examiner: Krsek-Staples; Julie
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis, L.L.P.
Claims
I claim:
1. A vaccinal pharmaceutical composition intended for preventing a
Neisseria meningitidis infection, which comprises, as therapeutic agents,
at least a first and a second molecule capable of binding to human
transferrin which are either a human transferrin receptor of N.
meningitidis or a subunit thereof; the said first molecule originating
from a first strain of N. meningitidis which possesses a human transferrin
receptor comprising a high molecular weight subunit and a lower molecular
weight subunit, and in which the lower molecular weight subunit is
recognised by an antiserum to the receptor of N. meningitidis strain 2394
(receptor 2394) and is not recognised by an antiserum to the receptor of
N. meningitidis strain 2169 (receptor 2169); and the said second molecule
originating from a second strain of N. meningitidis which possesses a
human transferrin receptor comprising a high molecular weight subunit and
a lower molecular weight subunit, and in which the lower molecular weight
subunit is recognised by an anti-receptor 2169 antiserum and is not
recognised by an anti-receptor 2394 antiserum.
2. A vaccinal pharmaceutical composition according to claim 1, which
comprises, as therapeutic agents, at least a first and a second molecule
capable of binding human transferrin; the said first molecule originating
from a first strain of N. meningitidis which possesses a human transferrin
receptor in which the high molecular subunit weight and the lower
molecular weight subunit are recognised by an anti-receptor 2394
antiserum; and the said second molecule originating from a second strain
of N. meningitidis which possesses a human transferrin receptor in which
the high molecular weight subunit and the lower molecular weight subunit
are recognised by an anti-receptor 2169 antiserum.
3. A vaccinal pharmaceutical composition according to claims 1 or 2, which
comprises, as therapeutic agents, at least a first and a second molecule
capable of binding to human transferrin; the said first molecule
originating from a first strain of N. meningitidis which possesses a human
transferrin receptor comprising a subunit of high molecular weight of 100
kD approximately to 90 kD and a subunit of lower molecular weight of 75 kD
to 60 kD; and the said second molecule originating from a second strain of
N. meningitidis which possesses a human transferrin receptor comprising of
a subunit of high molecular weight of 100 kD approximately to 90 kD and a
subunit of lower molecular weight of 90 kD to 80 kD.
4. A vaccinal pharmaceutical composition according to claim 3, in which the
said first molecule originates from a first strain of N. meningitidis
which possesses a human transferrin receptor comprising a subunit of high
molecular weight of 93-95 kD approximately and a subunit of lower
molecular weight of 72 kD to 65 kD.
5. A vaccinal pharmaceutical composition according to claim 4, in which the
said first molecule originates from a first strain of N. meningitidis
which possesses a human transferrin receptor comprising a subunit of high
molecular weight of 93 kD approximately and a subunit of lower molecular
weight of 67-70 kD approximately.
6. A vaccinal pharmaceutical composition according to claim 5, in which the
said second molecule originates from a second strain of N. meningitidis
which possesses a human transferrin receptor comprising a subunit of high
molecular weight of 100 kD approximately to 95 kD and a subunit of lower
molecular weight of 87 kD to 85 kD.
7. A vaccinal pharmaceutical composition according to claim 6, in which the
said second molecule originates from a second strain of N. meningitidis
which possesses a human transferrin receptor comprising a subunit of high
molecular weight of 98 kD approximately and a subunit of lower molecular
weight of 87 kD approximately.
8. A vaccinal pharmaceutical composition according to claim 1, in which the
said first and second molecules originate respectively from a first and
second strain of N. meningitidis serogroup B.
9. A vaccinal pharmaceutical composition for preventing a Neisseria
meningitidis infection, which comprises therapeutic agents comprising a
first and a second molecule capable of binding to human transferrin which
are either a human transferrin receptor of N. meningitidis or a subunit
thereof; said first molecule originating from a first strain of N.
meningitidis which possesses a human transferrin receptor comprising a
high molecular weight subunit and a lower molecular weight subunit, and in
which the lower molecular weight subunit is recognized by an antiserum to
the receptor of N. meningitidis strain 2394 and is not recognized by an
antiserum to the receptor of N. meningitidis strain 2169, and said second
molecule, originating from a second strain of N. meningitidis which
possesses a human transferrin receptor comprising a high molecular weight
subunit and a lower molecular weight subunit, and in which the lower
molecular weight subunit is recognized by an antiserum to the receptor of
N. meningitidis strain 2169 and is not recognized by an antiserum to the
receptor of N. meningitidis strain 2394.
Description
The present invention relates to a vaccinal pharmaceutical composition
intended for the prevention of meningitis caused by Neisseria
meningitidis.
Generally speaking, meningitis is either of viral origin or of bacterial
origin. The bacteria mainly responsible are N. meningitidis and
Haemophilus influenzae, which are implicated, respectively, in
approximately 40 and 50% of cases of bacterial meningitis.
N. meningitidis accounts for approximately 600 to 800 cases of meningitis
per annum in France. In the USA, the number of cases amounts to
approximately 2,500 to 3,000 per annum.
The species N. meningitidis is subdivided into serogroups according to the
nature of the capsular polysaccharides. Although a dozen serogroups exist,
90% of cases of meningitis are attributable to 3 serogroups: A, B and C.
There are effective vaccines based on capsular polysaccharides to prevent
meningitis caused by N. meningitidis serogroups A and C. These
polysaccharides, as such, exhibit little or no immunogenicity in infants
under 2 years of age, and do not induce immune memory. However, these
drawbacks may be overcome by conjugating these polysaccharides to a
carrier protein.
On the other hand, the polysaccharide of N. meningitidis group B exhibits
little or no immunogenicity in man, either in conjugated or in
unconjugated form. Thus, it is seen to be highly desirable to seek a
vaccine against meningitis induced by N. meningitidis, in particular of
serogroup B, other than a vaccine based on polysaccharide.
To this end, various proteins of the outer membrane of N. meningitidis have
already been proposed. Special attention has focused on the membrane
receptor for human transferrin.
Generally speaking, the large majority of bacteria require iron for their
growth, and have developed specific systems for acquiring this metal. As
regards N. meningitidis in particular, which is a strict pathogen of man,
the iron can be abstracted only from human iron-transport proteins such as
transferrin and lactoferrin, since the amount of iron in free form is
negligible in man (of the order of 10.sup.-18 M), and in any case
insufficient to permit bacterial growth.
Thus, N. meningitidis possesses a human transferrin receptor and a human
lactoferrin receptor, which enable it to bind these iron-chelating
proteins and thereafter to take up the iron needed for its growth.
The transferrin receptor of N. meningitidis strain B16B6 has been purified
by Schryvers et al. (WO 90/12591) from a membrane extract. This protein as
purified evidently consists essentially of two types of polypeptide: a
polypeptide of high apparent molecular weight of 100 kD and a polypeptide
of lower apparent molecular weight of approximately 70 kD, as visualised
after polyacrylamide gel electrophoresis in the presence of SDS.
The product of the purification carried out, in particular, by Schryvers is
referred to, by arbitrary definition and for the requirements of the
present patent application, as the transferrin receptor, and the
polypeptides of which it consists are referred to as subunits. In the text
below, the subunits of high molecular weight and of lower molecular weight
are referred to as Tbp1 and Tbp2, respectively.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is an SDS-PAGE gel with 7.5% acrylamide, in which columns A and B
correspond to the receptors of N. meningitidis strains 2169 and 2394,
respectively. The arrows pointing horizontally indicate the position of
the reference proteins of known apparent molecular mass (94 kD,
phosphorylase B; 67 kD, albumin).
It has now been found that there are at least two types of strain which
differ in the constitution of their respective transferrin receptors. This
was demonstrated by studying membrane extracts of several tens of strains
of N. meningitidis of miscellaneous origins. These membrane extracts were
first subjected to SDS-PAGE gel electrophoresis and then
electrotransferred onto nitrocellulose sheets. These nitrocellulose sheets
were incubated:
a) in the presence of a rabbit antiserum directed towards the transferrin
receptor purified from N. meningitidis strain B16B6, also referred to as
2394;
b) in the presence of a rabbit antiserum directed towards the transferrin
receptor purified from N. meningitidis strain 2169; or
c) in the presence of human transferrin conjugated to peroxidase.
As regards a) and b), the recognition of the subunits of the transferrin
receptor is visualised by adding an anti-rabbit immunoglobulins antibody
coupled to peroxidase, followed by addition of the substrate for this
enzyme.
Tables I and II hereinbelow show the profile of some representative strains
as seen on SDS-PAGE gel containing 7.5% polyacrylamide; the bands are
characterised by their apparent molecular weight expressed in kilodaltons
(kD):
TABLE I
______________________________________
Strains
2394 (B; 2a;
P1.2:L2.3)
2228 (B;
nd) 2234 (Y; nd)
550 (C; 2a:)
2170 (B;
2154 (C; nd)
179 (C;
2a:P1.2:2:L3)
2248 (B; nd)
2a:P1.2)
______________________________________
Detection with anti-
93 93 99
receptor 2394 antiserum
68 69 69
Detection with anti-
93 93 99
receptor 2169 antiserum
Detection with
68 69 69
transferrin-peroxidase
______________________________________
NB: The symbols in brackets denote, in order, the serogroup, serotype,
subtype and immunotype.
TABLE II
__________________________________________________________________________
Strains
2169 132 1001
876 867
(B:9:
1000
1604
(C:15:
(A:4:
(B:19:
1951
2449
(B:2b:
P1.9)
(B:nd)
(B:nd)
P1.16)
P1.9)
P1.6)
(A:nd)
(B:nd)
P1.2)
__________________________________________________________________________
Detection with anti-receptor
96 98 98 98 98 96 94 94 93
2394 antiserum
Detection with anti-receptor
96 98 98 98 98 96 94 94 93
2169 antiserum
87 85 83 81 79 88 87 85 85
Detection with transferrin-
87 85 83 81 79 88 87 85 85
peroxidase
__________________________________________________________________________
NB: The symbols in brackets denote, in order, the serogroup, serotype,
subtype and immunotype.
The results appearing in the first 2 lines of the tables show that there
are two types of strains:
The first type (Table I) corresponds to strains which possess a receptor in
which both of the subunits are recognised by anti-receptor 2394 antiserum
while only the high molecular weight subunit is recognised by
anti-receptor 2169 antiserum.
The second type (Table II) corresponds to strains which possess a receptor
in which both of the subunits are recognised by anti-receptor 2169
antiserum while only the high molecular weight subunit is recognised by
anti-receptor 2394 antiserum.
Consequently, there exists an antigenic diversity in respect of the lower
molecular weight subunit. This diversity is, however, limited since it
resolves into 2 major types, in contrast to the suggestion made by
Griffiths et al., FEMS Microbiol. Lett. (1990) 69: 31.
[Moreover, it will be noted that, irrespective of the type of strain, the
subunit capable of binding to transferrin is always the lower molecular
weight subunit (Tables I and II, third line of results).]
In accordance with these findings, it had been tempting to conclude that an
effective vaccine against all N. meningitidis infections could be
adequately composed of a transferrin receptor or exclusively of its high
molecular weight subunit, irrespective of the strain of origin of the
receptor, since this subunit is recognised by both types of antiserum.
Surprisingly, this has now been found not to be the case, inasmuch as the
high molecular weight subunit does not appear to be capable of inducing
the production of neutralising type antibodies. Only the smaller of the 2
subunits of the receptor appears to be capable of fulfilling this
function. Since this lower molecular weight subunit is characterised by a
significant antigenic variation from the first type to the second type of
strain, a single type of transferrin receptor should not be sufficient for
vaccinating against all N. meningitidis infections.
Thus, the invention provides for:
i) A vaccinal pharmaceutical composition which comprises, as therapeutic
agents, at least a first and a second molecule capable of binding to human
transferrin; the said first molecule originating from a first strain of N.
meningitidis which possesses a human transferrin receptor at least
consisting of a high molecular weight subunit (Tbp1) and a lower molecular
weight subunit (Tbp2), and in which the lower molecular weight subunit
(Tbp2) is recognised by an antiserum to the receptor of N. meningitidis
strain 2394 (receptor 2394) and is not recognised by an antiserum to the
receptor of N. meningitidis strain 2169 (receptor 2169); and the said
second molecule originating from a second strain of N. meningitidis which
possesses a human transferrin receptor at least consisting of a high
molecular weight subunit (Tbp1) and a lower molecular weight subunit
(Tbp2), and in which the lower molecular weight subunit (Tbp2) is
recognised by an anti-receptor 2169 antiserum and is not recognised by an
anti-receptor 2394 antiserum;
ii) A vaccination kit containing:
a) A pharmaceutical composition which comprises, as therapeutic agent, at
least a first molecule capable of binding to human transferrin; the said
first molecule originating from a first strain of N. meningitidis which
possesses a human transferrin receptor at least consisting of a high
molecular weight subunit and a lower molecular weight subunit, and in
which the lower molecular weight subunit is recognised by an antiserum to
the receptor of N. meningitidis strain 2394 (receptor 2394) and is not
recognised by an antiserum to the receptor of N. meningisidis strain 2169
(receptor 2169);
b) A pharmaceutical composition which comprises, as therapeutic agent, at
least a second molecule capable of binding to human transferrin; the said
second molecule originating from a second strain of N. meningitidis which
possesses a human transferrin receptor at least consisting of a high
molecular weight subunit and a lower molecular weight subunit, and in
which the lower molecular weight subunit is recognised by an anti-receptor
2169 antiserum and is not recognised by an anti-receptor 2394 antiserum;
and
c) Instructions for the concomitant or consecutive administration of the
compositions a) and b);
iii) The combined therapeutic use of at least a first and a second molecule
capable of binding to human transferrin; the said first molecule
originating from a first strain of N. meningitidis which possesses a human
transferrin receptor at least consisting of a high molecular weight
subunit and a lower molecular weight subunit, and in which the lower
molecular weight subunit is recognised by an antiserum to the receptor of
N. meningitidis strain 2394 (receptor 2394) and is not recognised by an
antiserum to the receptor of N. meningisidis strain 2169 (receptor 2169);
and the said second molecule originating from a second strain of N.
meningitidis which possesses a human transferrin receptor at least
consisting of a high molecular weight subunit and a lower molecular weight
subunit, and in which the lower molecular weight subunit is recognised by
an anti-receptor 2169 antiserum and is not recognised by an anti-receptor
2394 antiserum; and
iv) A method of vaccination against N. meningitidis infections, which
comprises the act of administering an effective amount, from a therapeutic
standpoint, of at least a first and a second molecule capable of binding
to human transferrin, concomitantly or consecutively, to a subject
requiring such a vaccinal treatment; the said first molecule originating
from a first strain of N. meningitidis which possesses a human transferrin
receptor at least consisting of a high molecular weight subunit and a
lower molecular weight subunit, and in which the lower molecular weight
subunit is recognised by an antiserum to the receptor of N. meningitidis
strain 2394 (receptor 2394) and is not recognised by an antiserum to the
receptor of N. meningitidis strain 2169 (receptor 2169); and the said
second molecule originating from a second strain of N. meningitidis which
possesses a human transferrin receptor at least consisting of a high
molecular weight subunit and a lower molecular weight subunit, and in
which the lower molecular weight subunit is recognised by an anti-receptor
2169 antiserum and is not recognised by an anti-receptor 2394 antiserum.
"Molecule capable of binding to human transferrin" is understood to mean
either a human transferrin receptor originating from N. meningitidis (that
is to say a molecule comprising, in particular, 2 types of subunit), or
exclusively the subunit of the receptor, capable of binding to human
transferrin, as well as a fragment or an analogue of this subunit.
A transferrin receptor may be obtained in purified form from a strain of N.
meningitidis previously cultured in a medium deficient in iron in free
form, in particular according to the method of Schryvers et al., WO
90/12591, described in a similar manner in Schryvers et al., Infect.
Immun. (1988) 56 (5):1144. Alternatively, a transferrin receptor
originating from a strain of N. meningitidis may be produced by employing
genetic engineering techniques. The DNA fragment or fragments coding for
the subunits of the receptor may be expressed jointly or separately in a
heterologous expression system (e.g. bacterium, yeast, mammalian cell).
The subunits, in free form or associated in the form of the receptor, are,
in this case, collected from a culture and purified. When the subunits are
produced in this way in free form, provision may be made for reassociating
them in the form of the receptor by subjecting them to a suitable
treatment.
The subunit capable of binding to human transferrin (lower molecular weight
subunit) may be obtained in purified form (that is to say dissociated and
isolated from the high molecular weight subunit), in particular, from a
receptor purified according to the method of Schryvers et al., by
subjecting the receptor to the action of a strongly denaturing agent such
as 8M urea or 6M guanidine HCl, and then separating the dissociated
subunits by standard chromatographic methods such as ion exchange
chromatography or gel filtration. Alternatively, the subunit may be
produced according to genetic engineering methods. These methods are, in
addition, entirely suited to the production of fragments or analogues of
the subunit.
As an example, the subunits Tbp1 and Tbp2 of the strains 2394 and 2169 are
described by reference to their amino acid sequences as shown in the
sequence identifiers Nos. 1 to 4 (SEQ ID No. 1 to 4).
"Fragment of the subunit capable of binding to human transferrin" is
understood to mean a peptide having an amino acid sequence which is
included in the sequence of the subunit. "Analogue of the subunit capable
of binding to human transferrin" is understood to mean a protein having an
amino acid sequence which exhibits an at least 80%, preferably at least
90% and, as an absolute preference, at least 95% homology with the
sequence of the subunit. For the purposes of the present invention, it
should be clearly understood that such a fragment or such an analogue must
retain the immunogenic properties of the subunit.
N. meningitidis strains 2394 (B:2a:P1.2:L2.3) and 2169 (B:9:P1.9:L3.7),
which are commonly used in laboratories, are available to the public from
the Collection of the Pasteur Institute, 25 rue du Dr Roux 75015 Paris,
under the respective registration numbers CIP 7908 and CIP 7917.
Furthermore, the anti-receptor antisera which are required in order to
distinguish the strains of N. meningitidis may be obtained as follows:
A receptor is first purified from an initial strain (2394 or 2169)
according to the method of Schryvers et al. Albino New Zealand rabbits
receive subcutaneously and intramuscularly 100 .mu.g of the receptor in
the presence of Freund's complete adjuvant. 21 days and 42 days after the
first injection, the rabbits again receive 100 .mu.g of the purified
receptor, but on these occasions in the presence of Freund's incomplete
adjuvant. 15 days after the last injection, the animals' serum is
withdrawn, then decomplemented and filtered through a membrane of porosity
0.45 .mu.m. The filtrate is thereafter exhaustively extracted by contact
with the initial strain which, to this end, has been cultured beforehand
in the presence of iron (under these conditions, synthesis of the
transferrin receptor is repressed). The contacting procedure is as
follows: 10 ml of the filtrate are added to 10.sup.10 cfu (colony forming
units) of a culture of the initial strain. Adsorption is carried out
overnight at 4.degree. C. with agitation. The bacteria are then removed by
centrifugation. The supernatant is recovered and then subjected again to 2
successive adsorption operations as described above.
The type of a strain (with respect to the nature of its transferrin
receptor) may be identified from membrane extracts derived from cultures
deficient in iron in free form, employing conventional techniques such as
SDS-PAGE gel electrophoresis, followed by immunoblotting using an
antiserum such as that described above.
The first molecule participating in the vaccinal composition originates
from a first strain of N. meningitidis which possesses a transferrin
receptor essentially consisting of (i) a subunit of high molecular weight,
advantageously of 100 to 90 kD and preferably of 93-95 kD approximately,
and (ii) a subunit of lower molecular weight, advantageously of 75 to 60
kD and preferably of 72 to 65 kD, and, as an absolute preference,
respectively (i) of 93 kD and (ii) of 67-70 kD approximately.
The second molecule participating in the vaccinal composition originates
from a second strain of N. meningitidis which possesses a transferrin
receptor essentially consisting of (i) a subunit of high molecular weight,
advantageously of 100 to 90 kD, preferably of 100 to 95 kD and, as an
absolute preference, of 98 kD approximately, and (ii) a subunit of lower
molecular weight, advantageously of 90 to 80 kD, preferably of 87 to 85 kD
and, as an absolute preference, of 87 kD approximately.
The molecular weights stated above are apparent molecular weights as
visualised after electrophoresis of a receptor purified on SDS-PAGE gel.
Such an electrophoresis may be carried out according to the method of
Laemmli illustrated as follows:
A polyacrylamide gel (16 cm.times.20 cm.times.1 mm in thickness) comprising
a 5% pregel and a 7.5% separating gel is first prepared in electrophoresis
buffer (Tris 6 g/l, glycine 28.8 g/l, 0.1% SDS).
In addition, 50 .mu.l of sample buffer (62 mM Tris-HCl pH 6.8, 2% SDS, 5%
.beta.-mercaptoethanol, 1% glycerol, 0.001% bromophenol blue) are added to
50 .mu.l of a solution of purified receptor at a concentration of 0.6
mg/ml (in 50 mM phosphate buffer pH 8.0 containing 0.05% Sarkosyl). The
mixture is incubated for 5 min in a boiling water bath. 17 .mu.l
(equivalent to 5 .mu.g of protein) of the sample thus prepared are placed
in a well in the gel. A sample prepared in a similar manner containing
molecular weight markers is added in parallel. Electrophoresis is carried
out in electrophoresis buffer at 50 volts for 15 hours. The gel is fixed
and stained with Coomassie blue.
Generally speaking, the first or the second molecule which is useful for
the purposes of the present invention can originate from a strain of N.
meningitidis of any serogroup. Advantageously, the first or the second
molecule originates from a strain of N. meningitidis serogroup B.
Preferably, the first and second molecules originate respectively from a
first and a second strain of N. meningitidis serogroup B.
According to an absolutely preferred aspect of the invention, the first
molecule originates from the strain 2394 while the second molecule
originates from the strain 2169.
A pharmaceutical composition according to the invention may be manufactured
in a conventional manner. In particular, the therapeutic agent or agents
according to the invention is/are combined with a diluent or vehicle which
is acceptable from a pharmaceutical standpoint. A composition according to
the invention may be administered by any conventional route in use in the
vaccine field, especially subcutaneously, intramuscularly or
intravenously, for example in the form of an injectable suspension. The
administration can take place in a single dose or in a dose repeated one
or several times after a certain time interval. The appropriate dosage
varies in accordance with various parameters, for example with the
individual being treated or with the mode of administration.
The invention is described in greater detail in the examples below and with
reference to FIG. 1, which illustrates an electrophoresis on SDS-PAGE gel
with 7.5% polyacrylamide, in which the columns A and B correspond to the
receptors of N. meningitidis strains 2169 and 2394, respectively. The
arrows pointing horizontally indicate the position of the reference
proteins of known apparent molecular mass (94 kD, phosphorylase B; 67 kD,
albumin).
EXAMPLE 1
Purification of the Transferrin Receptor from the Strain 2394
1A--Culture
A lyophilisate of N. meningitidis strain 2394 is taken up in approximately
1 ml of Mueller-Hinton broth (MHB, Difco). The bacterial suspension is
then plated out on Muller-Hinton solid medium containing cooked blood
(5%).
After 24 h of incubation at 37.degree. C. in an atmosphere containing 10%
of CO.sub.2, the bacterial lawn is collected in order to inoculate 150 ml
of MHB pH 7.2, distributed in three 250-ml Erlenmeyers. Incubation is
carried out for 3 h at 37.degree. C. with agitation. Each of the 3
cultures so produced permits the inoculation of 400 ml of MHB pH 7.2
supplemented with 30 .mu.M of ethylenediaminedi(o-hydroxyphenylacetic
acid) (EDDA, Sigma), which is a chelating agent for iron in free form.
After 16 h of culture at 37.degree. C. with agitation, the cultures are
monitored for their purity by microscopic observation after Gram staining.
The suspension is centrifuged and the pellet containing the microbes is
weighed and stored at -20.degree. C.
1B--Purification
The purification method is essentially that described by Schryvers et al.
(supra).
The bacterial pellet obtained in 1A is thawed and then resuspended in 200
ml of 50 mM Tris-HCl buffer, pH 8.0 (buffer A). The suspension is
centrifuged for 20 min at 15,000 x g at 4.degree. C. The pellet is
recovered and then resuspended in buffer A at a final concentration of 150
g/l. 150-ml fractions are treated for 8 min at 800 bars in a cell lyser
working under high pressure (Rannie, model 8.30H). The cell lysate thereby
obtained is centrifuged for 15 min at 4.degree. C. at 15,000 x g. The
supernatant is recovered and then centrifuged for 75 min at 4.degree. C.
at 200,000 x g.
After removal of the supernatant, the pellet is taken up in buffer A and,
after protein assay by the Lowry method, the concentration of the
suspension is adjusted to 5 mg/ml.
1.75 mg of biotinylated human transferrin are then added to 1.4 ml of the
membrane suspension according to the method described by Schryvers. The
final concentration of the membrane fraction is 4 mg/ml. The mixture is
incubated for 1 hour at 37.degree. C. and then centrifuged at 100,000 x g
for 75 minutes at 4.degree. C. The membrane pellet is taken up with buffer
A containing 0.1M NaCl, and incubated for 60 min at room temperature.
After solubilisation, a certain volume of 30% (w/v) N-LauroylSarkosine and
of 500 mM EDTA are added to this suspension so that the final
concentrations of Sarkosyl and EDTA are 0.5% and 5 mM, respectively. After
incubation for 15 min at 37.degree. C. with agitation, 1 ml of
streptavidin-agarose resin (Pierce), previously washed in buffer A, is
added. The suspension is incubated for 15 min at room temperature and then
centrifuged at 1,000 x g for 10 min. The resin is then packed in a column
and the direct eluate is discarded.
The resin is washed with 3 column volumes of 50 mM Tris-HCl buffer pH 8.0
containing 1M NaCl, 10 mM EDTA, 0.5% Sarkosyl (buffer B), and then with
one column volume of buffer B containing 750 mM guanidine HCl. The
transferrin receptor is then eluted with buffer B containing 2M guanidine
HCl, 0.05% Sarkosyl. The eluate is collected in fractions whose volume
corresponds to 1 Vol. in Lubes containing 1 Vol. of 50 mM Tris-HCl pH 8.0,
1M NaCl. The optical density of the eluate at 280 nm is measured at the
column outlet using a UV detector.
The fractions corresponding to the elution peak are collected, dialysed
against 10 mM phosphate buffer, pH 8.0 containing 0.05% Sarkosyl and
lyophilised. The lyophilisate is taken up in water at a 10-fold higher
concentration. The solution is dialysed a second time against 50 mM
phosphate buffer pH 8.0 containing 0.05% Sarkosyl (buffer C), and the
solution is then filtered through a membrane of porosity 0.22 .mu.m.
The protein content is determined and adjusted to 1 mg/ml by adding buffer
C, under aseptic conditions. This preparation is stored at -70.degree. C.
EXAMPLE 2
Purification of the Transferrin Receptor from the Strain 2169
Culturing of the strain 2169 and purification of the transferrin receptor
are performed under conditions identical to those described in Example 1.
EXAMPLE 3
Vaccinal Pharmaceutical Composition Intended for Preventing N. meningitidis
Infections
The sterile solutions obtained in Examples 1 and 2 are thawed. In order to
prepare one liter of vaccine containing 100 .mu.g/ml of each of the active
principles, the following solutions are mixed under sterile conditions:
______________________________________
Solution of receptor 2394 at a
100 ml
concentration of 1 mg/ml in buffer C
Solution of receptor 2169 at a
100 ml
concentration of 1 mg/ml in buffer C
Buffered physiological saline (PBS),
300 ml
pH 6.0
Aluminium hydroxide containing 10 mg
50 ml
Al.sup.+++ /ml
Merthiolate, 1% (w/v) in PBS
10 ml
PBS qs 1,000 ml
______________________________________
EXAMPLE 4
Demonstration of the Importance of the Lower Molecular Weight Subunit as a
Vaccinal Agent
Albino New Zealand rabbits receive subcutaneously and intramuscularly 100
.mu.g of the receptor 2394 or 2169 (as obtained in Example 1 or 2), in the
presence of Freund's complete adjuvant. 21 and 42 days after the first
injection, the rabbits again receive 100 .mu.g of the purified receptor,
but this time in the presence of Freund's incomplete adjuvant. 15 days
after the last injection, the animals' serum is withdrawn, then
decomplemented and filtered through a membrane of porosity of 0.45 .mu.m.
The filtrate is thereafter exhaustively extracted by contact with the
initial strain (2394 or 2169) which, to this end, has been cultured
beforehand in the presence of iron in free form (under these conditions,
synthesis of the transferrin receptor is repressed). The contacting
procedure is as follows: 10 ml of the filtrate are added to 10.sup.10 cfu
(colony forming units) of a culture of the initial strain. Adsorption is
carried out overnight at 4.degree. C. with agitation. The bacteria are
then removed by centrifugation. The supernatant is recovered and then
subjected again to 2 successive adsorption operations as described above.
A dilution series of each of the antisera, anti-receptor 2394 and
anti-receptor 2169, is prepared in M199 medium (Gibco). 200 .mu.l of each
dilution are placed in the wells of a microtitration plate (8.times.12
in.). A control test is carried out with 200 .mu.l of M199 medium. Into
each of the wells there are added (i) 100 .mu.l of a culture in the
exponential growth phase of a strain of N. meningitidis, in Mueller-Hinton
medium supplemented with 30 .mu.M EDDA and (ii) 100 .mu.l of complement
(young rabbit serum, diluted).
After 30 min of incubation at 37.degree. C. with gentle agitation, 1 ml of
Mueller-Hinton medium containing 1 ml of Noble agar in the supercooled
state is added into each well. After solidification of the medium,
incubation is carried out for 18-24 hours at 37.degree. C.; the number of
colony forming units in each well is then evaluated. The reciprocal of the
final dilution of antiserum in the presence of which a 50% lysis is
observed relative to the control corresponds to the bactericidal titre.
The results are presented in Table III below:
______________________________________
Bactericidal activity
Rabbit No. 1 Rabbit No. 2
Serum before
Anti- Serum before
Anti-
2394 receptor 2169 receptor
immunisation
antiserum immunisation
antiserum
______________________________________
2394 <8 2048 <8 <8
2228 <8 1024 <8 <8
2154 <8 2048 <8 <8
2234 <8 2048 <8 <8
2448 <8 256 <8 <4
2169 <16 <16 <8 1024
896 <8 <8 <8 65
______________________________________
The anti-receptor 2394 antiserum has bactericidal activity exclusively
against strains of the first type as defined in the present application
(2394, 2228, 2154, 2234 and 2448), while the anti-receptor 2169 antiserum
has bactericidal activity exclusively against strains of the second type
(2169 and 876). This strongly suggests that the production of neutralising
antibodies is essentially induced by the lower molecular weight subunit
which carries the antigenic variability.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 4
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 579 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Neisseria meningitidis 2394 subunit Tbp2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CysLeuGlyGlyGlyGlySerPheAspLeuAspSerValGluThrVal
151015
GlnAspMetHisSerLysProLysTyrGluAspGluLysSerGlnPro
202530
GluSerGlnGlnAspValSerGluAsnSerGlyAlaAlaTyrGlyPhe
354045
AlaValLysLeuProArgArgAsnAlaHisPheAsnProLysTyrLys
505560
GluLysHisLysProLeuGlySerMetAspTrpLysLysLeuGlnArg
65707580
GlyGluProAsnSerPheSerGluArgAspGluLeuGluLysLysArg
859095
GlySerSerGluLeuIleGluSerLysTrpGluAspGlyGlnSerArg
100105110
ValValGlyTyrThrAsnPheThrTyrValArgSerGlyTyrValTyr
115120125
LeuAsnLysAsnAsnIleAspIleLysAsnAsnIleValLeuPheGly
130135140
ProAspGlyTyrLeuTyrTyrLysGlyLysGluProSerLysGluLeu
145150155160
ProSerGluLysIleThrTyrLysGlyThrTrpAspTyrValThrAsp
165170175
AlaMetGluLysGlnArgPheGluGlyLeuGlySerAlaAlaGlyGly
180185190
AspLysSerGlyAlaLeuSerAlaLeuGluGluGlyValLeuArgAsn
195200205
GlnAlaGluAlaSerSerGlyHisThrAspPheGlyMetThrSerGlu
210215220
PheGluValAspPheSerAspLysThrIleLysGlyThrLeuTyrArg
225230235240
AsnAsnArgIleThrGlnAsnAsnSerGluAsnLysGlnIleLysThr
245250255
ThrArgTyrThrIleGlnAlaThrLeuHisGlyAsnArgPheLysGly
260265270
LysAlaLeuAlaAlaAspLysGlyAlaThrAsnGlySerHisProPhe
275280285
IleSerAspSerAspSerLeuGluGlyGlyPheTyrGlyProLysGly
290295300
GluGluLeuAlaGlyLysPheLeuSerAsnAspAsnLysValAlaAla
305310315320
ValPheGlyAlaLysGlnLysAspLysLysAspGlyGluAsnAlaAla
325330335
GlyProAlaThrGluThrValIleAspAlaTyrArgIleThrGlyGlu
340345350
GluPheLysLysGluGlnIleAspSerPheGlyAspValLysLysLeu
355360365
LeuValAspGlyValGluLeuSerLeuLeuProSerGluGlyAsnLys
370375380
AlaAlaPheGlnHisGluIleGluGlnAsnGlyValLysAlaThrVal
385390395400
CysCysSerAsnLeuAspTyrMetSerPheGlyLysLeuSerLysGlu
405410415
AsnLysAspAspMetPheLeuGlnGlyValArgThrProValSerAsp
420425430
ValAlaAlaArgThrGluAlaLysTyrArgGlyThrGlyThrTrpTyr
435440445
GlyTyrIleAlaAsnGlyThrSerTrpSerGlyGluAlaSerAsnGln
450455460
GluGlyGlyAsnArgAlaGluPheAspValAspPheSerThrLysLys
465470475480
IleSerGlyThrLeuThrAlaLysAspArgThrSerProAlaPheThr
485490495
IleThrAlaMetIleLysAspAsnGlyPheSerGlyValAlaLysThr
500505510
GlyGluAsnGlyPheAlaLeuAspProGlnAsnThrGlyAsnSerHis
515520525
TyrThrHisIleGluAlaThrValSerGlyGlyPheTyrGlyLysAsn
530535540
AlaIleGluMetGlyGlySerPheSerPheProGlyAsnAlaProGlu
545550555560
GlyLysGlnGluLysAlaSerValValPheGlyAlaLysArgGlnGln
565570575
LeuValGln
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 884 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Neisseria meningitidis 2394 subunit Tbp1.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GluAsnValGlnAlaGluGlnAlaGlnGluLysGlnLeuAspThrIle
151015
GlnValLysAlaLysLysGlnLysThrArgArgAspAsnGluValThr
202530
GlyLeuGlyLysLeuValLysSerSerAspThrLeuSerLysGluGln
354045
ValLeuAsnIleArgAspLeuThrArgTyrAspProGlyIleAlaVal
505560
ValGluGlnGlyArgGlyAlaSerSerGlyTyrSerIleArgGlyMet
65707580
AspLysAsnArgValSerLeuThrValAspGlyValSerGlnIleGln
859095
SerTyrThrAlaGlnAlaAlaLeuGlyGlyThrArgThrAlaGlySer
100105110
SerGlyAlaIleAsnGluIleGluTyrGluAsnValLysAlaValGlu
115120125
IleSerLysGlySerAsnSerSerGluTyrGlyAsnGlyAlaLeuAla
130135140
GlySerValAlaPheGlnThrLysThrAlaAlaAspIleIleGlyGlu
145150155160
GlyLysGlnTrpGlyIleGlnSerLysThrAlaTyrSerGlyLysAsp
165170175
HisAlaLeuThrGlnSerLeuAlaLeuAlaGlyArgSerGlyGlyAla
180185190
GluAlaLeuLeuIleTyrThrLysArgArgGlyArgGluIleHisAla
195200205
HisLysAspAlaGlyLysGlyValGlnSerPheAsnArgLeuValLeu
210215220
AspGluAspLysLysGluGlyGlySerGlnTyrArgTyrPheIleVal
225230235240
GluGluGluCysHisAsnGlyTyrAlaAlaCysLysAsnLysLeuLys
245250255
GluAspAlaSerValLysAspGluArgLysThrValSerThrGlnAsp
260265270
TyrThrGlySerAsnArgLeuLeuAlaAsnProLeuGluTyrGlySer
275280285
GlnSerTrpLeuPheArgProGlyTrpHisLeuAspAsnArgHisTyr
290295300
ValGlyAlaValLeuGluArgThrGlnGlnThrPheAspThrArgAsp
305310315320
MetThrValProAlaTyrPheThrSerGluAspTyrValProGlySer
325330335
LeuLysGlyLeuGlyLysTyrSerGlyAspAsnLysAlaGluArgLeu
340345350
PheValGlnGlyGluGlySerThrLeuGlnGlyIleGlyTyrGlyThr
355360365
GlyValPheTyrAspGluArgHisThrLysAsnArgTyrGlyValGlu
370375380
TyrValTyrHisAsnAlaAspLysAspThrTrpAlaAspTyrAlaArg
385390395400
LeuSerTyrAspArgGlnGlyIleAspLeuAspAsnArgLeuGlnGln
405410415
ThrHisCysSerHisAspGlySerAspLysAsnCysArgProAspGly
420425430
AsnLysProTyrSerPheTyrLysSerAspArgMetIleTyrGluGlu
435440445
SerArgAsnLeuPheGlnAlaValPheLysLysAlaPheAspThrAla
450455460
LysIleArgHisAsnLeuSerIleAsnLeuGlyTyrAspArgPheLys
465470475480
SerGlnLeuSerHisSerAspTyrTyrLeuGlnAsnAlaValGlnAla
485490495
TyrAspLeuIleThrProLysLysProProPheProAsnGlySerLys
500505510
AspAsnProTyrArgValSerIleGlyLysThrThrValAsnThrSer
515520525
ProIleCysArgPheGlyAsnAsnThrTyrThrAspCysThrProArg
530535540
AsnIleGlyGlyAsnGlyTyrTyrAlaAlaValGlnAspAsnValArg
545550555560
LeuGlyArgTrpAlaAspValGlyAlaGlyIleArgTyrAspTyrArg
565570575
SerThrHisSerGluAspLysSerValSerThrGlyThrHisArgAsn
580585590
LeuSerTrpAsnAlaGlyValValLeuLysProPheThrTrpMetAsp
595600605
LeuThrTyrArgAlaSerThrGlyPheArgLeuProSerPheAlaGlu
610615620
MetTyrGlyTrpArgAlaGlyGluSerLeuLysThrLeuAspLeuLys
625630635640
ProGluLysSerPheAsnArgGluAlaGlyIleValPheLysGlyAsp
645650655
PheGlyAsnLeuGluAlaSerTyrPheAsnAsnAlaTyrArgAspLeu
660665670
IleAlaPheGlyTyrGluThrArgThrGlnAsnGlyGlnThrSerAla
675680685
SerGlyAspProGlyTyrArgAsnAlaGlnAsnAlaArgIleAlaGly
690695700
IleAsnIleLeuGlyLysIleAspTrpHisGlyValTrpGlyGlyLeu
705710715720
ProAspGlyLeuTyrSerThrLeuAlaTyrAsnArgIleLysValLys
725730735
AspAlaAspIleArgAlaAspArgThrPheValThrSerTyrLeuPhe
740745750
AspAlaValGlnProSerArgTyrValLeuGlyLeuGlyTyrAspHis
755760765
ProAspGlyIleTrpGlyIleAsnThrMetPheThrTyrSerLysAla
770775780
LysSerValAspGluLeuLeuGlySerGlnAlaLeuLeuAsnGlyAsn
785790795800
AlaAsnAlaLysLysAlaAlaSerArgArgThrArgProTrpTyrVal
805810815
ThrAspValSerGlyTyrTyrAsnIleLysLysHisLeuThrLeuArg
820825830
AlaGlyValTyrAsnLeuLeuAsnTyrArgTyrValThrTrpGluAsn
835840845
ValArgGlnThrAlaGlyGlyAlaValAsnGlnHisLysAsnValGly
850855860
ValTyrAsnArgTyrAlaAlaProGlyArgAsnTyrThrPheSerLeu
865870875880
GluMetLysPhe
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 887 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Neisseria meningitidis 2169 subunit Tbp1.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GluAsnValGlnAlaGlyGlnAlaGlnGluLysGlnLeuAspThrIle
151015
GlnValLysAlaLysLysGlnLysThrArgArgAspAsnGluValThr
202530
GlyLeuGlyLysLeuValLysThrAlaAspThrLeuSerLysGluGln
354045
ValLeuAspIleArgAspLeuThrArgTyrAspProGlyIleAlaVal
505560
ValGluGlnGlyArgGlyAlaSerSerGlyTyrSerIleArgGlyMet
65707580
AspLysAsnArgValSerLeuThrValAspGlyLeuAlaGlnIleGln
859095
SerTyrThrAlaGlnAlaAlaLeuGlyGlyThrArgThrAlaGlySer
100105110
SerGlyAlaIleAsnGluIleGluTyrGluAsnValLysAlaValGlu
115120125
IleSerLysGlySerAsnSerValGluGlnGlySerGlyAlaLeuAla
130135140
GlySerValAlaPheGlnTyrLysThrAlaAspAspValIleGlyGlu
145150155160
GlyArgGlnTrpGlyIleGlnSerLysThrAlaTyrSerGlyLysAsn
165170175
ArgGlyLeuThrGlnSerIleAlaLeuAlaGlyArgIleGlyGlyAla
180185190
GluAlaLeuLeuIleHisThrGlyArgArgAlaGlyGluIleArgAla
195200205
HisGluAspAlaGlyArgGlyValGlnSerPheAsnArgLeuValPro
210215220
ValGluAspSerSerGluTyrAlaTyrPheIleValGluAspGluCys
225230235240
GluGlyLysAsnTyrGluThrCysLysSerLysProLysLysAspVal
245250255
ValGlyLysAspGluArgGlnThrValSerThrArgAspTyrThrGly
260265270
ProAsnArgPheLeuAlaAspProLeuSerTyrGluSerArgSerTrp
275280285
LeuPheArgProGlyPheArgPheGluAsnLysArgHisTyrIleGly
290295300
GlyIleLeuGluHisThrGlnGlnThrPheAspThrArgAspMetThr
305310315320
ValProAlaPheLeuThrLysAlaValPheAspAlaAsnSerLysGln
325330335
AlaGlySerLeuProGlyAsnGlyLysTyrAlaGlyAsnHisLysTyr
340345350
GlyGlyLeuPheThrAsnGlyGluAsnGlyAlaLeuValGlyAlaGlu
355360365
TyrGlyThrGlyValPheTyrAspGluThrHisThrLysSerArgTyr
370375380
GlyLeuGluTyrValTyrThrAsnAlaAspLysAspThrTrpAlaAsp
385390395400
TyrAlaArgLeuSerTyrAspArgGlnGlyIleGlyLeuAspAsnHis
405410415
PheGlnGlnThrHisCysSerAlaAspGlySerAspLysTyrCysArg
420425430
ProSerAlaAspLysProPheSerTyrTyrLysSerAspArgValIle
435440445
TyrGlyGluSerHisArgLeuLeuGlnAlaAlaPheLysLysSerPhe
450455460
AspThrAlaLysIleArgHisAsnLeuSerValAsnLeuGlyPheAsp
465470475480
ArgPheAspSerAsnLeuArgHisGlnAspTyrTyrTyrGlnHisAla
485490495
AsnArgAlaTyrSerSerLysThrProProLysThrAlaAsnProAsn
500505510
GlyAspLysSerLysProTyrTrpValSerIleGlyGlyGlyAsnVal
515520525
ValThrGlyGlnIleCysLeuPheGlyAsnAsnThrTyrThrAspCys
530535540
ThrProArgSerIleAsnGlyLysSerTyrTyrAlaAlaValArgAsp
545550555560
AsnValArgLeuGlyArgTrpAlaAspValGlyAlaGlyLeuArgTyr
565570575
AspTyrArgSerThrHisSerAspAspGlySerValSerThrGlyThr
580585590
HisArgThrLeuSerTrpAsnAlaGlyIleValLeuLysProAlaAsp
595600605
TrpLeuAspLeuThrTyrArgThrSerThrGlyPheArgLeuProSer
610615620
PheAlaGluMetTyrGlyTrpArgSerGlyValGlnSerLysAlaVal
625630635640
LysIleAspProGluLysSerPheAsnLysGluAlaGlyIleValPhe
645650655
LysGlyAspPheGlyAsnLeuGluAlaSerTrpPheAsnAsnAlaTyr
660665670
ArgAspLeuIleValArgGlyTyrGluAlaGlnIleLysAsnGlyLys
675680685
GluGluAlaLysGlyAspProAlaTyrLeuAsnAlaGlnSerAlaArg
690695700
IleThrGlyIleAsnIleLeuGlyLysIleAspTrpAsnGlyValTrp
705710715720
AspLysLeuProGluGlyTrpTyrSerThrPheAlaTyrAsnArgVal
725730735
HisValArgAspIleLysLysArgAlaAspArgThrAspIleGlnSer
740745750
HisLeuPheAspAlaIleGlnProSerArgTyrValValGlyLeuGly
755760765
TyrAspGlnProGluGlyLysTrpGlyValAsnGlyMetLeuThrTyr
770775780
SerLysAlaLysGluIleThrGluLeuLeuGlySerArgAlaLeuLeu
785790795800
AsnGlyAsnSerArgAsnThrLysAlaThrAlaArgArgThrArgPro
805810815
TrpTyrIleValAspValSerGlyTyrTyrThrIleLysLysHisPhe
820825830
ThrLeuArgAlaGlyValTyrAsnLeuLeuAsnTyrArgTyrValThr
835840845
TrpGluAsnValArgGlnThrAlaGlyGlyAlaValAsnGlnHisLys
850855860
AsnValGlyValTyrAsnArgTyrAlaAlaProGlyArgAsnTyrThr
865870875880
PheSerLeuGluMetLysPhe
885
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Neisseria meningitidis 2169 subunit Tbp2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CysLeuGlyGlyGlyGlySerPheAspLeuAspSerValAspThrGlu
151015
AlaProArgProAlaProLysTyrGlnAspValSerSerGluLysPro
202530
GlnAlaGlnLysAspGlnGlyGlyTyrGlyPheAlaMetArgLeuLys
354045
ArgArgAsnTrpTyrProGlyAlaGluGluSerGluValLysLeuAsn
505560
GluSerAspTrpGluAlaThrGlyLeuProThrLysProLysGluLeu
65707580
ProLysArgGlnLysSerValIleGluLysValGluThrAspGlyAsp
859095
SerAspIleTyrSerSerProTyrLeuThrProSerAsnHisGlnAsn
100105110
GlySerAlaGlyAsnGlyValAsnGlnProLysAsnGlnAlaThrGly
115120125
HisGluAsnPheGlnTyrValTyrSerGlyTrpPheTyrLysHisAla
130135140
AlaSerGluLysAspPheSerAsnLysLysIleLysSerGlyAspAsp
145150155160
GlyTyrIlePheTyrHisGlyGluLysProSerArgGlnLeuProAla
165170175
SerGlyLysValIleTyrLysGlyValTrpHisPheValThrAspThr
180185190
LysLysGlyGlnAspPheArgGluIleIleGlnProSerLysLysGln
195200205
GlyAspArgTyrSerGlyPheSerGlyAspGlySerGluGluTyrSer
210215220
AsnLysAsnGluSerThrLeuLysAspAspHisGluGlyTyrGlyPhe
225230235240
ThrSerAsnLeuGluValAspPheGlyAsnLysLysLeuThrGlyLys
245250255
LeuIleArgAsnAsnAlaSerLeuAsnAsnAsnThrAsnAsnAspLys
260265270
HisThrThrGlnTyrTyrSerLeuAspAlaGlnIleThrGlyAsnArg
275280285
PheAsnGlyThrAlaThrAlaThrAspLysLysGluAsnGluThrLys
290295300
LeuHisProPheValSerAspSerSerSerLeuSerGlyGlyPhePhe
305310315320
GlyProGlnGlyGluGluLeuGlyPheArgPheLeuSerAspAspGln
325330335
LysValAlaValValGlySerAlaLysThrLysAspLysLeuGluAsn
340345350
GlyAlaAlaAlaSerGlySerThrGlyAlaAlaAlaSerGlyGlyAla
355360365
AlaGlyThrSerSerGluAsnSerLysLeuThrThrValLeuAspAla
370375380
ValGluLeuThrLeuAsnAspLysLysIleLysAsnLeuAspAsnPhe
385390395400
SerAsnAlaAlaGlnLeuValValAspGlyIleMetIleProLeuLeu
405410415
ProLysAspSerGluSerGlyAsnThrGlnAlaAspLysGlyLysAsn
420425430
GlyGlyThrGluPheThrArgLysPheGluHisThrProGluSerAsp
435440445
LysLysAspAlaGlnAlaGlyThrGlnThrAsnGlyAlaGlnThrAla
450455460
SerAsnThrAlaGlyAspThrAsnGlyLysThrLysThrTyrGluVal
465470475480
GluValCysCysSerAsnLeuAsnTyrLeuLysTyrGlyMetLeuThr
485490495
ArgLysAsnSerLysSerAlaMetGlnAlaGlyGlyAsnSerSerGln
500505510
AlaAspAlaLysThrGluGlnValGluGlnSerMetPheLeuGlnGly
515520525
GluArgThrAspGluLysGluIleProThrAspGlnAsnValValTyr
530535540
ArgGlySerTrpTyrGlyHisIleAlaAsnGlyThrSerTrpSerGly
545550555560
AsnAlaSerAspLysGluGlyGlyAsnArgAlaGluPheThrValAsn
565570575
PheAlaAspLysLysIleThrGlyLysLeuThrAlaGluAsnArgGln
580585590
AlaGlnThrPheThrIleGluGlyMetIleGlnGlyAsnGlyPheGlu
595600605
GlyThrAlaLysThrAlaGluSerGlyPheAspLeuAspGlnLysAsn
610615620
ThrThrArgThrProLysAlaTyrIleThrAspAlaLysValLysGly
625630635640
GlyPheTyrGlyProLysAlaGluGluLeuGlyGlyTrpPheAlaTyr
645650655
ProGlyAspLysGlnThrGluLysAlaThrAlaThrSerSerAspGly
660665670
AsnSerAlaSerSerAlaThrValValPheGlyAlaLysArgGlnGln
675680685
ProValGln
690
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